The following information is presented solely to assist the understanding of the reader. None of the information is admitted to describe prior art to the claims of the present invention.
Gene therapy is a major area of research in drug development. Gene therapy has been considered a desirable mechanism to correct genetically determined diseases resulting from the failure to produce certain proteins and acquired diseases such as autoimmunity and cancer. One example of a class of genetically determined diseases that are considered amenable to gene therapy is hemophilia. Hemophilia B, for example, is a bleeding disorder that results from the absence of functional blood clotting Factor IX (“F.IX”). The disease state is classified as severe, moderate or mild, depending on the level of functional F.IX. (Lusher, J. M. (1999) Thromb Haemost 82:572-5751). Approximately 5,200 males are afflicted with the disease in the U.S. with approximately 45% of these cases being of the severe type. In severe cases of hemophilia B (<1% of normal F.IX levels) there are frequent bleeding events that can be life threatening and often produce debilitating destruction of the patient's joints. The current therapy for hemophilia B is the administration of F.IX protein in response to bleeding events only. The use of either blood derived or recombinant F.IX has shown that tremendous clinical and quality of life benefits can be achieved by converting the most severe hemophilia B cases into the moderate or mild range. In some countries F.IX protein is given prophylactically in the most severe cases, despite the fact that these treatments are extremely expensive (Ljung, R. C. (1999) Thromb Haemost 82:525-530). The prophylactic use of F.IX is not frequent in the U.S.
Gene therapy could provide a new prophylactic approach for the treatment of diseases such as hemophilia B. A technological barrier to commercialization of gene therapy, however, is the need for practical, effective and safe gene delivery methods. In animal models of hemophilia, viral-based vectors have been used successfully to administer the human F.IX gene either to liver or muscle. (Kay, M. A., et al. (1993) Science 262:117-119; Herzog, R. W., et al. (1999) Nat Med: 56-63; Snyder, R. O., et al. (1999) Nat Med 5:64-70; Chao, H., et al. (1999) Gene Ther 6:1695-1704; Lozier, J. N., et al. (1999) Blood 94:3968-3975; Kaufman, R. J. (1999) Hum Gene Ther 10:2091-2107). In some cases, these approaches have led to long-term (>2 years) expression of therapeutic levels of F.IX in a canine model of hemophilia B (Herzog, R. W., et al. (1999) Nat Med 5:56-63). However, the limitations of viral-based approaches have been extensively reported. For instance, re-administration is not possible with these vectors because of the humoral immune response generated against the viral proteins. In addition to manufacturing challenges to obtain adequate reproducible vector supply, there are also significant safety concerns associated with viral vectors, particularly for those targeting the liver for gene expression. Not withstanding the problems associated with viral gene therapy, viruses have been considered by many to be more efficient than non-viral delivery vehicles.
A problem of non-viral gene therapy is to achieve the delivery and expression of sufficient nucleic acid to result in a tangible, physiologically relevant expression. Although DNA plasmids in isotonic saline (so-called ‘naked’ DNA) were shown several years ago to transfect a variety of cells in vivo, the lack of stability of such unprotected plasmids to enzymatic degradation is associated with irreproducibility in uptake leading to highly variable expression and biological responses in animal models. The very low bioavailability of ‘naked’ plasmid in most tissues also requires high doses of plasmids to be administered to generate a pharmacological response.
The field of non-viral gene delivery has therefore been directed to the development of more efficient synthetic delivery systems able to increase the efficiency of plasmid delivery, confer prolonged expression and provide for storage stable formulations as is expected of other pharmaceutical formulations.
To overcome the problem of degradation of nucleic acids, typically plasmid DNA (“pDNA”), and enhance the efficiency of gene transfection, cationic condensing agents (such as polybrene, dendrimers, chitosan, lipids, and peptides) have been developed to protect pDNA by condensing it through electrostatic interaction. (A. P. Rolland, From genes to gene medicines: recent advances in nonviral gene delivery, review in Therapeutic drug carrier systems, 15(2):143-198 (1998).) However, the use of condensed plasmid particles for transfection of a large number of muscle cells in vivo has not been successful as compared directly to “naked” DNA. Wolff, J. A., et al., J. Cell Sci., 103, 1249, 1992. In particular, due to the physiology of the muscle, the use of rigid condensed particles containing plasmid for efficient transfection of a larger number of muscle cells has not been successful to date because cationic lipid and polylysine plasmid complexes do not cross the external lamina to gain access to the caveolae and T tubules. Id.
Additional strategies that include the modulation of the plasmid surface charge and hydrophobicity by interaction with protective, interactive non-condensing systems (e.g., PINC™ polymers) have shown advantages over the use of ‘naked’ DNA for direct administration to solid tissues. [WO9621470, U.S. Pat. No. 6,040,295, incorporated herein by reference.]
Biodegradable microspheres have also been used in gene delivery that encapsulate the nucleic acid. For example, WO0078357, Chen, W. et al, disclosed matrices, films, gels and hydrogels which include hyaluronic acid (HA) derivatized with a dihydrazide and crosslinked to a nucleic acid forming slow release microspheres. WO9524929, Boekelheide, K. et al., disclosed encapsulation of genes in a matrix preferably in the form of a microparticle such as a microsphere, microcapsule, a film, an implant, or a coating on a device such as a stent. U.S. Pat. No. 6,048,551, Beer, S. et al. disclosed a controlled release gene delivery system utilizing poly (lactide-co-glycolide) (PLGA), hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, and the Ludragit R, L, and E series of polymers and copolymer microspheres to encapsulate the gene vector. Luo D et al. Pharm Res 1999 Aug. 16(8):1300-8, reported the characterization of systems for controlled delivery of DNA from implantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate)) and injectable microspheres (PLGA and PLA: poly (D, L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively). Despite their promise, microspheres can pose manufacturing difficulties and can adversely constrain the release of DNA in vivo, particularly in muscle tissue.
Thus, despite these recent advances, there remains a need for additional and improved formulated nucleic acid compositions and methods of administering the same for gene therapy.